Methods of treating cancer using PD-L1 axis binding antagonists and VEGF antagonists

ABSTRACT

The present invention describes combination treatment comprising a PD-1 axis binding antagonist, chemotherapy and optionally a VEGF antagonist and methods for use thereof, including methods of treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a continuation of International Patent ApplicationNo. PCT/US2013/043452, filed on May 30, 2013, which claims the benefitfrom U.S. Provisional Application No. 61/653,861, filed on 31 May 2012,which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 17, 2014, isnamed P4926R1C1SeqListing.txt and is 14,299 bytes in size.

BACKGROUND OF THE INVENTION

The provision of two distinct signals to T-cells is a widely acceptedmodel for lymphocyte activation of resting T lymphocytes byantigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp. Biol.Med. ScL 53: 27-42 (1975). This model further provides for thediscrimination of self from non-self and immune tolerance. Bretscher etal, Science 169: 1042-1049 (1970); Bretscher, P. A., P.N.A.S. USA 96:185-190 (1999); Jenkins et al, J. Exp. Med. 165: 302-319 (1987). Theprimary signal, or antigen specific signal, is transduced through theT-cell receptor (TCR) following recognition of foreign antigen peptidepresented in the context of the major histocompatibility-complex (MHC).The second or co-stimulatory signal is delivered to T-cells byco-stimulatory molecules expressed on antigen-presenting cells (APCs),and induce T-cells to promote clonal expansion, cytokine secretion andeffector function. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). Inthe absence of co-stimulation, T-cells can become refractory to antigenstimulation, do not mount an effective immune response, and further mayresult in exhaustion or tolerance to foreign antigens.

In the two-signal model T-cells receive both positive and negativesecondary co-stimulatory signals. The regulation of such positive andnegative signals is critical to maximize the host's protective immuneresponses, while maintaining immune tolerance and preventingautoimmunity. Negative secondary signals seem necessary for induction ofT-cell tolerance, while positive signals promote T-cell activation.While the simple two-signal model still provides a valid explanation fornaive lymphocytes, a host's immune response is a dynamic process, andco-stimulatory signals can also be provided to antigen-exposed T-cells.The mechanism of co-stimulation is of therapeutic interest because themanipulation of co-stimulatory signals has shown to provide a means toeither enhance or terminate cell-based immune response. Recently, it hasbeen discovered that T cell dysfunction or anergy occurs concurrentlywith an induced and sustained expression of the inhibitory receptor,programmed death 1 polypeptide (PD-1). As a result, therapeutictargeting of PD-1 and other molecules which signal through interactionswith PD-1, such as programmed death ligand 1 (PD-L1) and programmeddeath ligand 2 (PD-L2) are an area of intense interest.

PD-L1 is overexpressed in many cancers and is often associated with poorprognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, the majority oftumor infiltrating T lymphocytes predominantly express PD-1, in contrastto T lymphocytes in normal tissues and peripheral blood T lymphocytesindicating that up-regulation of PD-1 on tumor-reactive T cells cancontribute to impaired antitumor immune responses (Blood 2009114(8):1537). This may be due to exploitation of PD-L1 signalingmediated by PD-L1 expressing tumor cells interacting with PD-1expressing T cells to result in attenuation of T cell activation andevasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir M Eet al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of thePD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing oftumors.

The inhibition of PD-1 axis signaling through its direct ligands (e.g.,PD-L1, PD-L2) has been proposed as a means to enhance T cell immunityfor the treatment of cancer (e.g., tumor immunity). Moreover, similarenhancements to T cell immunity have been observed by inhibiting thebinding of PD-L1 to the binding partner B7-1. Optimal therapeutictreatment could combine blockade of PD-1 receptor/ligand interactionwith other anti-cancer agents. There remains a need for such an optimaltherapy for treating, stabilizing, preventing, and/or delayingdevelopment of various cancers.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a combination treatment comprisingoxaliplatin, leucovorin and 5-FU and a PD-1 axis binding antagonist withor without a VEGF antagonist.

Provided herein are methods for treating cancer or slowing progressionof cancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and oxaliplatin,leucovorin and 5-FU. In some aspects the method further comprisesadministering a VEGF antagonist.

The cancer may be a melanoma, a colorectal cancer, a non-small cell lungcancer, an ovarian cancer, a breast cancer, a prostate cancer, apancreatic cancer, hematological malignancy or a renal cell carcinoma.The cancer may be at early stage or at late stage. In some embodiments,the subject treated is a human.

In some embodiments, the treatment results in sustained response in theindividual after cessation of the treatment. In some embodiments, thetreatment produces a complete response, a partial response, or stabledisease in the subject.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist, a PD-L1 binding antagonist or a PD-L2 binding antagonist. Insome embodiments, the PD-1 binding antagonist inhibits binding of PD-1to PD-L1 and/or binding of PD-1 to PD-L2. In some embodiments, the PD-1binding antagonist is an antibody (e.g., antibody MDX-1106, CT-011 andMerck 3745 described herein), an antigen binding fragments thereof, animmunoadhesin, a fusion protein, or an oligopeptide. In someembodiments, the PD-L1 binding antagonist inhibits binding of PD-L1 toPD-1 and/or binding of PD-L1 to B7-1. In some embodiments, the PD-L1binding antagonist is an antibody (e.g., antibody YW243.55.570 andMDX-1105 described herein), an antigen binding fragments thereof, animmunoadhesin, a fusion protein, or an oligopeptide. In someembodiments, the PD-L2 binding antagonist inhibits binding of PD-L2 toPD-1. In some embodiments, the PD-L2 binding antagonist is an antibody,an antigen binding fragments thereof, an immunoadhesin (e.g., AMP-224described herein), a fusion protein, or an oligopeptide.

In some embodiments, the VEGF antagonist is an antibody, e.g., amonoclonal antibody. In some embodiments the anti-VEGF antibody bindsthe same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709. The anti-VEGF antibody may be a humanizedantibody or a human antibody. In some embodiments the anti-VEGF antibodyis bevacizumab. In some embodiments the anti-VEGF antibody has a heavychain variable region comprising the following amino acid sequence:

(SEQ ID NO: 22) EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYFDVWGQGTLVT VSSand a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 23) DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYFTSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR.

In another aspect, provided is a kit comprising a PD-1 axis bindingantagonist, oxaliplatin, leucovorin and 5-FU with or without a VEGFantagonist for treating or delaying progression of a cancer in anindividual or enhancing immune function in an individual having cancer.The kit may comprise a PD-1 axis binding antagonist and a package insertcomprising instructions for using the PD-1 axis binding antagonist incombination with oxaliplatin, leucovorin and 5-FU with or without a VEGFantagonist to treat or delay progression of cancer in an individual, orenhancing immune function in an individual having cancer. The kit maycomprise a VEGF antagonist and a package insert comprising instructionsfor using the VEGF antagonist in combination with a PD-1 axis bindingantagonist and oxaliplatin, leucovorin and 5-FU to treat or delayprogression of cancer in an individual, or to enhance immune function inan individual having cancer. The kit may comprise a PD-1 axis bindingantagonist and a VEGF antagonist, and a package insert comprisinginstructions for using the PD-1 axis binding antagonist and the VEGFantagonist to treat or delay progression of cancer in an individual, orto enhance immune function in an individual having cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting changes in tumor volume with anti-PD-L1antibodies and FOLFOX co-treatment. The data demonstrate a significantreduction of tumor growth and sustained anti-tumor effect as compared toanti-PD-L1 antibodies or FOLFOX treatment alone.

FIG. 2 is a graph showing changes in body weight for the treatmentgroups shown in FIG. 1.

FIG. 3 is a graph depicting changes in tumor volume with anti-PD-L1antibodies in combination with FOLFOX as compared to anti-PD-L1antibodies in combination with FOLFOX and anti-VEGF antibody. The datademonstrate that additional administration of anti-VEGF antibodysignificantly reduced tumor growth and resulted in a sustainedanti-tumor effect as compared to treatment with anti-PD-L1 antibodies incombination with FOLFOX.

FIG. 4 is a graph showing changes in body weight for the treatmentgroups shown in FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and AnimalCell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J. B. LippincottCompany, 1993).

II. Definitions

The term “PD-1 axis binding antagonist” is a molecule that inhibits theinteraction of a PD-1 axis binding partner with either one or more ofits binding partner, so as to remove T-cell dysfunction resulting fromsignaling on the PD-1 signaling axis—with a result being to restore orenhance T-cell function. As used herein, a PD-1 axis binding antagonistincludes a PD-1 binding antagonist, a PD-L1 binding antagonist and aPD-L2 binding antagonist.

The term “PD-1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to itsbinding partners. In a specific aspect, the PD-1 binding antagonistinhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1binding antagonists include anti-PD-1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-1 withPD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reducesthe negative co-stimulatory signal mediated by or through cell surfaceproteins expressed on T lymphocytes mediated signaling through PD-1 soas render a dysfunctional T-cell less non-dysfunctional. In someembodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In aspecific aspect, a PD-1 binding antagonist is MDX-1106 described herein.In another specific aspect, a PD-1 binding antagonist is Merck 3745described herein. In another specific aspect, a PD-1 binding antagonistis CT-011 described herein.

The term “PD-L1 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as rendera dysfunctional T-cell less non-dysfunctional. In some embodiments, aPD-L1 binding antagonist is an anti-PD-L1 antibody. In a specificaspect, an anti-PD-L1 antibody is YW243.55.S70 described herein. Inanother specific aspect, an anti-PD-L1 antibody is MDX-1105 describedherein.

The term “PD-L2 binding antagonists” is a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to itsbinding partners. In a specific aspect, the PD-L2 binding antagonistinhibits binding of PD-L2 to PD-1. In some embodiments, the PD-L2antagonists include anti-PD-L2 antibodies, antigen binding fragmentsthereof, immunoadhesins, fusion proteins, oligopeptides and othermolecules that decrease, block, inhibit, abrogate or interfere withsignal transduction resulting from the interaction of PD-L2 with eitherone or more of its binding partners, such as PD-1. In one embodiment, aPD-L2 binding antagonist reduces the negative co-stimulatory signalmediated by or through cell surface proteins expressed on T lymphocytesmediated signaling through PD-L2 so as render a dysfunctional T-cellless non-dysfunctional. In some embodiments, a PD-L2 binding antagonistis a PD-L2 immunoadhesin. In a specific aspect, a PD-L2 immunoadhesin isAMP-224 described herein.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including its binding to one or more VEGF receptors. VEGFantagonists include anti-VEGF antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives which bind specifically toVEGF thereby sequestering its binding to one or more receptors,anti-VEGF receptor antibodies and VEGF receptor antagonists such assmall molecule inhibitors of the VEGFR tyrosine kinases.

The term “VEGF” or “VEGF-A” is used to refer to the 165-amino acid humanvascular endothelial cell growth factor and related 121-, 145-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by, e.g., Leung et al. Science, 246:1306 (1989), and Houck etal. Mol. Endocrin., 5:1806 (1991), together with the naturally occurringallelic and processed forms thereof. VEGF-A is part of a gene familyincluding VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-Aprimarily binds to two high affinity receptor tyrosine kinases, VEGFR-1(Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the major transmitterof vascular endothelial cell mitogenic signals of VEGF-A. Additionally,neuropilin-1 has been identified as a receptor for heparin-bindingVEGF-A isoforms, and may play a role in vascular development. The term“VEGF” or “VEGF-A” also refers to VEGFs from non-human species such asmouse, rat, or primate. Sometimes the VEGF from a specific species isindicated by terms such as hVEGF for human VEGF or mVEGF for murineVEGF. The term “VEGF” is also used to refer to truncated forms orfragments of the polypeptide comprising amino acids 8 to 109 or 1 to 109of the 165-amino acid human vascular endothelial cell growth factor.Reference to any such forms of VEGF may be identified in the presentapplication, e.g., by “VEGF (8-109),” “VEGF (1-109)” or “VEGF165.” Theamino acid positions for a “truncated” native VEGF are numbered asindicated in the native VEGF sequence. For example, amino acid position17 (methionine) in truncated native VEGF is also position 17(methionine) in native VEGF. The truncated native VEGF has bindingaffinity for the KDR and Flt-1 receptors comparable to native VEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. The antibody selected will normallyhave a binding affinity for VEGF, for example, the antibody may bindhVEGF with a Kd value of between 100 nM-1 pM. Antibody affinities may bedetermined by a surface plasmon resonance based assay (such as theBIAcore assay as described in PCT Application Publication No.WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); andcompetition assays (e.g. RIA's), for example. In certain embodiments,the anti-VEGF antibody of the invention can be used as a therapeuticagent in targeting and interfering with diseases or conditions whereinthe VEGF activity is involved. Also, the antibody may be subjected toother biological activity assays, e.g., in order to evaluate itseffectiveness as a therapeutic. Such assays are known in the art anddepend on the target antigen and intended use for the antibody. Examplesinclude the HUVEC inhibition assay; tumor cell growth inhibition assays(as described in WO 89/06692, for example); antibody-dependent cellularcytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays(U.S. Pat. No. 5,500,362); and agonistic activity or hematopoiesisassays (see WO 95/27062). An anti-VEGF antibody will usually not bind toother VEGF homologues such as VEGF-B or VEGF-C, nor other growth factorssuch as PlGF, PDGF or bFGF.

A “chimeric VEGF receptor protein” is a VEGF receptor molecule havingamino acid sequences derived from at least two different proteins, atleast one of which is as VEGF receptor protein. In certain embodiments,the chimeric VEGF receptor protein is capable of binding to andinhibiting the biological activity of VEGF.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as defined above,e.g., antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptoror Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec™ (ImatinibMesylate). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar,Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenictherapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine5:1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) (e.g.,Table 2 listing known antiangiogenic factors); and Sato. Int. J. Clin.Oncol., 8:200-206 (2003) (e.g., Table 1 lists anti-angiogenic agentsused in clinical trials).

The term “dysfunction” in the context of immune dysfunction, refers to astate of immune reduced responsiveness to antigenic stimulation. Theterm includes the common elements of both exhaustion and/or anergy inwhich antigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viralor pathogen clearance) relative to such levels before the intervention.In one embodiment, the level of enhancement is as least 50%,alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner ofmeasuring this enhancement is known to one of ordinary skill in the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenecity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include treatmentwith anti-PDL antibodies oxaliplatin, leucovorin and 5-FU with orwithout a VEGF antagonist.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies(e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and c isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, and in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler andMilstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (ColdSpring Harbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhuet al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and technologies for producing human or human-likeantibodies in animals that have parts or all of the human immunoglobulinloci or genes encoding human immunoglobulin sequences (see, e.g., WO1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits etal., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33(1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851(1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg andHuszar, Intern. Rev. Immunol. 13: 65-93 (1995).

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof. In some cases, the intact antibody may have one ormore effector functions.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fc region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855(1984)). Chimeric antibodies of interest herein include PRIMATIZED®antibodies wherein the antigen-binding region of the antibody is derivedfrom an antibody produced by, e.g., immunizing macaque monkeys with anantigen of interest. As used herein, “humanized antibody” is used asubset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-human species (donor antibody) such as mouse, rat, rabbitor non-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

A “human consensus framework” or “acceptor human framework” is aframework that represents the most commonly occurring amino acidresidues in a selection of human immunoglobulin VL or VH frameworksequences. Generally, the selection of human immunoglobulin VL or VHsequences is from a subgroup of variable domain sequences. Generally,the subgroup of sequences is a subgroup as in Kabat et al., Sequences ofProteins of Immunological Interest, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Examples includefor the VL, the subgroup may be subgroup kappa I, kappa II, kappa III orkappa IV as in Kabat et al., supra. Additionally, for the VH, thesubgroup may be subgroup I, subgroup II, or subgroup III as in Kabat etal., supra. Alternatively, a human consensus framework can be derivedfrom the above in which particular residues, such as when a humanframework residue is selected based on its homology to the donorframework by aligning the donor framework sequence with a collection ofvarious human framework sequences. An acceptor human framework “derivedfrom” a human immunoglobulin framework or a human consensus frameworkmay comprise the same amino acid sequence thereof, or it may containpre-existing amino acid sequence changes. In some embodiments, thenumber of pre-existing amino acid changes are 10 or less, 9 or less, 8or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 orless.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al., supra. In one embodiment, the VH subgroup III consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 30) EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1), (SEQ ID NO: 31)WVRQAPGKGLEWV (HC-FR2),, SEQ ID NO: 32 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(HC-FR3),, (SEQ ID NO: 33) WGQGTLVTVSA (HC-FR4),.

A “VL kappa I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al., supra. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITC (LC-FR1), (SEQ ID NO: 35)WYQQKPGKAPKLLIY (LC-FR2), (SEQ ID NO: 36)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3), (SEQ ID NO: 37) FGQGTKVEIKR(LC-FR4).

An “amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al., Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis of HVR and/or framework residues is described by, forexample: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994);Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds to a target has a dissociation constant(Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certainembodiments, an antibody specifically binds to an epitope on a proteinthat is conserved among the protein from different species. In anotherembodiment, specific binding can include, but does not require exclusivebinding.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA(including IgA-1 and IgA-2), IgE, IgD or IgM. The Ig fusions preferablyinclude the substitution of a domain of a polypeptide or antibodydescribed herein in the place of at least one variable region within anIg molecule. In a particularly preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgG1 molecule. For the production of immunoglobulinfusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995. Forexample, useful immunoadhesins as second medicaments useful forcombination therapy herein include polypeptides that comprise theextracellular or PD-1 binding portions of PD-L1 or PD-L2 or theextracellular or PD-L1 or PD-L2 binding portions of PD-1, fused to aconstant domain of an immunoglobulin sequence, such as a PD-L1 ECD-Fc, aPD-L2 ECD-Fc, and a PD-1 ECD-Fc, respectively. Immunoadhesincombinations of Ig Fc and ECD of cell surface receptors are sometimestermed soluble receptors.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker but are in reading frame with each other.

A “PD-1 oligopeptide,” “PD-L1 oligopeptide,” or “PD-L2 oligopeptide” isan oligopeptide that binds, preferably specifically, to a PD-1, PD-L1 orPD-L2 negative costimulatory polypeptide, respectively, including areceptor, ligand or signaling component, respectively, as describedherein. Such oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Such oligopeptides are usually at least about 5amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100amino acids in length or more. Such oligopeptides may be identifiedusing well known techniques. In this regard, it is noted that techniquesfor screening oligopeptide libraries for oligopeptides that are capableof specifically binding to a polypeptide target are well known in theart (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT PublicationNos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. Proc. Natl. Acad. Sci. USA, 87:6378 (1990); Lowman, H. B. et al.Biochemistry, 30:10832 (1991); Clackson, T. et al. Nature, 352: 624(1991); Marks, J. D. et al., J. Mol. Biol., 222:581 (1991); Kang, A. S.et al. Proc. Natl. Acad. Sci. USA, 88:8363 (1991), and Smith, G. P.,Current Opin. Biotechnol., 2:668 (1991).

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Forexample, a VEGF-specific antagonist antibody binds VEGF and inhibits theability of VEGF to induce vascular endothelial cell proliferation or toinduce vascular permeability. The anti-PD-L1 antibodies of the inventionblock the signaling through PD-1 so as to restore a functional responseby T-cells from a dysfunctional state to antigen stimulation.

An “agonist” or activating antibody is one that enhances or initiatessignaling by the antigen to which it binds. In some embodiments, agonistantibodies cause or activate signaling without the presence of thenatural ligand.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2A,IgG2B), IgG3 and IgG4.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors, FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see M. Daëron, Annu.Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995).Other FcRs, including those to be identified in the future, areencompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997);Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton etal., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004/92219 (Hinton etal.). Binding to FcRn in vivo and serum half-life of human FcRnhigh-affinity binding polypeptides can be assayed, e.g., in transgenicmice or transfected human cell lines expressing human FcRn, or inprimates to which the polypeptides having a variant Fc region areadministered. WO 2004/42072 (Presta) describes antibody variants whichimproved or diminished binding to FcRs. See also, e.g., Shields et al.,J. Biol. Chem. 9(2): 6591-6604 (2001).

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, greater than about 10%, greaterthan about 20%, greater than about 30%, greater than about 40%, and/orgreater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with an antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., Kd values). The differencebetween said two values is, for example, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, and/or less thanabout 10% as a function of the reference/comparator value.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

An “effective amount” is at least the minimum concentration required toeffect a measurable improvement or prevention of a particular disorder.An effective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers as well as dormant tumors or micrometastatses.Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include squamous cell cancer, lung cancer (including small-celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer (includinggastrointestinal cancer), pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Preferably, the subject is a human. Patients are also subjects herein.

As used herein, “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; and “stable disease” or “SD”refers to neither sufficient shrinkage of target lesions to qualify forPR, nor sufficient increase to qualify for PD, taking as reference thesmallest SLD since the treatment started.

As used herein, “progressive disease” or “PD” refers to at least a 20%increase in the SLD of target lesions, taking as reference the smallestSLD recorded since the treatment started or the presence of one or morenew lesions.

As used herein, “progression free survival” (PFS) refers to the lengthof time during and after treatment during which the disease beingtreated (e.g., cancer) does not get worse. Progression-free survival mayinclude the amount of time patients have experienced a complete responseor a partial response, as well as the amount of time patients haveexperienced stable disease.

As used herein, “overall response rate” (ORR) refers to the sum ofcomplete response (CR) rate and partial response (PR) rate.

As used herein, “overall survival” refers to the percentage ofindividuals in a group who are likely to be alive after a particularduration of time.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan, and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; pemetrexed;callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesinsynthetic analogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral alpha-4integrin inhibitor; a sarcodictyin; spongistatin; nitrogen mustards suchas chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such asthe enediyne antibiotics (e. g., calicheamicin, especially calicheamicingamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al., Angew.Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®) and deoxydoxorubicin), epirubicin,esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine(XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acidanalogues such as denopterin, methotrexate, pteropterin, trimetrexate;purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine,thioguanine; pyrimidine analogs such as ancitabine, azacitidine,6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidinederivative), as well as other c-Kit inhibitors; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK®polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g.,paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin;leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluorometlhylornithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and toremifene (FARESTON®);anti-progesterones; estrogen receptor down-regulators (ERDs); estrogenreceptor antagonists such as fulvestrant (FASLODEX®); agents thatfunction to suppress or shut down the ovaries, for example, leutinizinghormone-releasing hormone (LHRH) agonists such as leuprolide acetate(LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate andtripterelin; anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole,vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).In addition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); anti-sense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1inhibitor (e.g., LURTOTECAN®); an anti-estrogen such as fulvestrant; aKit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinaseinhibitor); EGFR inhibitor such as erlotinib or cetuximab; an anti-VEGFinhibitor such as bevacizumab; irinotecan; rmRH (e.g., ABARELIX®);lapatinib and lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosinekinase small-molecule inhibitor also known as GW572016); 17AAG(geldanamycin derivative that is a heat shock protein (Hsp) 90 poison),and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

As used herein, the term “cytokine” refers generically to proteinsreleased by one cell population that act on another cell asintercellular mediators or have an autocrine effect on the cellsproducing the proteins. Examples of such cytokines include lymphokines,monokines; interleukins (“ILs”) such as IL-1, IL-la, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL10, IL-11, IL-12, IL-13, IL-15,IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including PROLEUKIN®rIL-2; a tumor-necrosis factor such as TNF-α or TNF-β, TGF-β1-3; andother polypeptide factors including leukemia inhibitory factor (“LIF”),ciliary neurotrophic factor (“CNTF”), CNTF-like cytokine (“CLC”),cardiotrophin (“CT”), and kit ligand (“KL”).

As used herein, the term “chemokine” refers to soluble factors (e.g.,cytokines) that have the ability to selectively induce chemotaxis andactivation of leukocytes. They also trigger processes of angiogenesis,inflammation, wound healing, and tumorigenesis. Example chemokinesinclude IL-8, a human homolog of murine keratinocyte chemoattractant(KC).

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

It is understood that aspects and variations of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand variations.

III. Methods

The methods of this invention may find use in treating conditions whereenhanced immunogenicity is desired such as increasing tumorimmunogenicity for the treatment of cancer. A variety of cancers may betreated, or their progression may be delayed.

In some embodiments, the individual has melanoma. The melanoma may be atearly stage or at late stage. In some embodiments, the individual hascolorectal cancer. The colorectal cancer may be at early stage or atlate stage. In some embodiments, the individual has non-small cell lungcancer. The non-small cell lung cancer may be at early stage or at latestage. In some embodiments, the individual has pancreatic cancer. Thepancreatic cancer may be at early stage or late state. In someembodiments, the individual has a hematological malignancy. Thehematological malignancy may be early stage or late stage. In someembodiments, the individual has ovarian cancer. The ovarian cancer maybe at early stage or at late stage. In some embodiments, the individualhas breast cancer. The breast cancer may be at early stage or at latestage. In some embodiments, the individual has renal cell carcinoma. Therenal cell carcinoma may be at early stage or at late stage.

In some embodiments, the subject treated is a human.

The combination therapy of the invention comprises administration of aPD-1 axis binding antagonist and oxaliplatin, leucovorin and 5-FU. Inanother aspect the invention provides a combination therapy comprisingthe administration of a PD-1 axis binding antagonist, a VEGF antagonistand oxaliplatin, leucovorin and 5-FU. The PD-1 axis binding antagonistand the VEGF antagonist may be administered in any suitable manner knownin the art. For example, The PD-1 axis binding antagonist and the VEGFantagonist may be administered sequentially (at different times) orconcurrently (at the same time).

In some embodiments, the methods of the invention may further compriseadministering an additional therapy. The additional therapy may beradiation therapy, surgery (e.g., lumpectomy and a mastectomy),chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy,immunotherapy, bone marrow transplantation, nanotherapy, monoclonalantibody therapy, or a combination of the foregoing. The additionaltherapy may be in the form of adjuvant or neoadjuvant therapy. In someembodiments, the additional therapy is the administration of smallmolecule enzymatic inhibitor or anti-metastatic agent. In someembodiments, the additional therapy is the administration of side-effectlimiting agents (e.g., agents intended to lessen the occurrence and/orseverity of side effects of treatment, such as anti-nausea agents,etc.). In some embodiments, the additional therapy is radiation therapy.In some embodiments, the additional therapy is surgery. In someembodiments, the additional therapy is a combination of radiationtherapy and surgery. The additional therapy may be one or more of thechemotherapeutic agents described hereabove.

Any of the PD-1 axis binding antagonists and the VEGF antagonistsdescribed below may be used in the methods of the invention.

PD-1 Axis Binding Antagonists

Provided herein is a method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist in combination withoxaliplatin, leucovorin and 5-FU with or without administration of aVEGF antagonist. For example, a PD-1 axis binding antagonist includes aPD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 bindingantagonist.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2.In another embodiment, a PD-L1 binding antagonist is a molecule thatinhibits the binding of PD-L1 to its binding partners. In a specificaspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiments, the PD-1 binding antagonist is selected from thegroup consisting of MDX-1106, Merck 3475 and CT-011. In someembodiments, the PD-L1 binding antagonist is selected from the groupconsisting of YW243.55.S70 and MDX-1105. In some embodiments, the PD-L2binding antagonist is AMP-224. MDX-1105, also known as BMS-936559, is ananti-PD-L1 antibody described in WO2007/005874. Antibody YW243.55.S70(SEQ ID No. 20) is an anti-PD-L1 described in WO 2010/077634 A1.MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is ananti-PD-1 antibody described in WO2006/121168. Merck 3745, also known asMK-3475 or SCH-900475, is an anti-PD-1 antibody described inWO2009/114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, isa PD-L2-Fc fusion soluble receptor described in WO2010/027827 andWO2011/066342.

Examples of anti-PD-L1 antibodies useful for the methods of thisinvention, and methods for making thereof are described in PCT patentapplication WO 2010/077634 A1, which are incorporated herein byreference.

In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1antibody. In some embodiments, the anti-PD-L1 antibody is capable ofinhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody.In some embodiments, the anti-PD-L1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is ahumanized antibody. In some embodiments, the anti-PD-L1 antibody is ahuman antibody.

The anti-PD-L1 antibodies useful in this invention, includingcompositions containing such antibodies, such as those described in WO2010/077634 A1, may be used in combination with oxaliplatin, leucovorin,5-FU with or without a VEGF antagonist to treat cancer.

In one embodiment, the anti-PD-L1 antibody contains a heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

(a) the HVR-H1 sequence is is (SEQ ID NO: 1) GFTFSX₁SWIH; (b) the HVR-H2sequence is (SEQ ID NO: 2) AWIX₂PYGGSX₃YYADSVKG; (c) the HVR-H3 sequenceis (SEQ ID NO: 3) RHWPGGFDY;

further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S.

In one specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,the polypeptide further comprises variable region heavy chain frameworksequences juxtaposed between the HVRs according to the formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

HC-FR1 is (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 is (SEQ ID NO:5) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 6)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 7) WGQGTLVTVSA.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(a) the HVR-L1 sequence is (SEQ ID NO: 8) RASQX₄X₅X₆TX₇X₈A; (b) theHVR-L2 sequence is (SEQ ID NO: 9) SASX₉LX₁₀S,; (c) the HVR-L3 sequenceis (SEQ ID NO: 10) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;

-   -   further wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is        A or F; X₈ is V or L; X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G,        F, or S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄        is H, V, P, T or I; X₁₅ is A, W, R, P or T.

In a still further aspect, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V;X₉ is F; X₁₀ is Y; X_(1i) is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A.In a still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the frameworksequences are VL kappa I consensus framework. In a still further aspect,at least one of the framework sequence is the following:

LC-FR1 is (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITC LC-FR2 is (SEQ ID NO:12) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 13)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 14) FGQGTKVEIKR.

In another embodiment, provided is an isolated anti-PD-L1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

-   -   (a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3,        wherein further:

(i) the HVR-H1 sequence is (SEQ ID NO: 1) GFTFSX₁SWIH; (ii) the HVR-H2sequence is (SEQ ID NO: 2) AWIX₂PYGGSX₃YYADSVKG (iii) the HVR-H3sequence is (SEQ ID NO: 3) RHWPGGFDY, and

-   -   (b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3,        wherein further:

(i) the HVR-L1 sequence is (SEQ ID NOs: 8) RASQX₄X₅X₆TX₇X₈A (ii) theHVR-L2 sequence is (SEQ ID NOs: 9) SASX₉LX₁₀S; and (iii) the HVR-L3sequence is (SEQ ID NOs: 10) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;

-   -   Further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S; X₄ is        D or V; X₅ is V or I; X₆ is S or N; X₇ is A or F; X₈ is V or L;        X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y,        F or W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V, P, T or I;        X₁₅ is A, W, R, P or T.

In a specific aspect, X₁ is D; X₂ is S and X₃ is T. In another aspect,X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁ isY; X₁₂ is L; X₁₃ is Y; X₁₄ is H; X₁₅ is A. In yet another aspect, X₁ isD; X₂ is S and X₃ is T, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H and X₁₅ is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 5)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 6) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 7) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 12)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 13) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 14) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

-   -   (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and        RHWPGGFDY (SEQ ID NO:3), respectively, or    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT        (SEQ ID NO:19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 5)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 6) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 7) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 12)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 13) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 14) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 20) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA,or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKR.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 5)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 6) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 7) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 12)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 13) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 14) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a still further embodiment, the invention provides for compositionscomprising any of the above described anti-PD-L1 antibodies incombination with at least one pharmaceutically-acceptable carrier.

In a still further embodiment, provided is an isolated nucleic acidencoding a light chain or a heavy chain variable region sequence of ananti-PD-L1 antibody, wherein:

-   -   (a) the heavy chain further comprises and HVR-H1, HVR-H2 and an        HVR-H3 sequence having at least 85% sequence identity to        GFTFSDSWIH (SEQ ID NO:15), AWISPYGGSTYYADSVKG (SEQ ID NO:16) and        RHWPGGFDY (SEQ ID NO:3), respectively, and    -   (b) the light chain further comprises an HVR-L1, HVR-L2 and an        HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:17), SASFLYS (SEQ ID NO:18) and QQYLYHPAT        (SEQ ID NO:19), respectively.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In aspect, theheavy chain variable region comprises one or more framework sequencesjuxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences is the following:

HC-FR1 (SEQ ID NO: 4) EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 5)WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 6) RFTISADTSKNTAYLQMNSLRAEDTAVYYCARHC-FR4 (SEQ ID NO: 7) WGQGTLVTVSA.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 12)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 13) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 14) FGQGTKVEIKR.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region if IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtheraspect, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a still further aspect, the nucleic acid further comprises a vectorsuitable for expression of the nucleic acid encoding any of thepreviously described anti-PD-L1 antibodies. In a still further specificaspect, the vector further comprises a host cell suitable for expressionof the nucleic acid. In a still further specific aspect, the host cellis a eukaryotic cell or a prokaryotic cell. In a still further specificaspect, the eukaryotic cell is a mammalian cell, such as Chinese HamsterOvary (CHO).

The anti-PD-L1 antibody or antigen binding fragment thereof, may be madeusing methods known in the art, for example, by a process comprisingculturing a host cell containing nucleic acid encoding any of thepreviously described anti-PD-L1 antibodies or antigen-binding fragmentin a form suitable for expression, under conditions suitable to producesuch antibody or fragment, and recovering the antibody or fragment.

In a still further embodiment, the invention provides for a compositioncomprising an anti-PD-L1 antibody or antigen binding fragment thereof asprovided herein and at least one pharmaceutically acceptable carrier.

VEGF Antagonists

The invention provides methods for treating cancer or slowingprogression of cancer in an individual comprising administering aneffective amount of a PD-1 pathway antagonist and a VEGF antagonist incombination with oxaliplatin, leucovorin and 5-FU. Any known VEGFantagonists are intended.

(i) VEGF Antigen

The VEGF antigen to be used for production of antibodies may be, e.g.,the VEGF₁₆₅ molecule as well as other isoforms of VEGF or a fragmentthereof containing the desired epitope. Other forms of VEGF useful forgenerating anti-VEGF antibodies of the invention will be apparent tothose skilled in the art.

Human VEGF was obtained by first screening a cDNA library prepared fromhuman cells, using bovine VEGF cDNA as a hybridization probe. Leung etal. (1989) Science, 246:1306. One cDNA identified thereby encodes a165-amino acid protein having greater than 95% homology to bovine VEGF;this 165-amino acid protein is typically referred to as human VEGF(hVEGF) or VEGF₁₆₅. The mitogenic activity of human VEGF was confirmedby expressing the human VEGF cDNA in mammalian host cells. Mediaconditioned by cells transfected with the human VEGF cDNA promoted theproliferation of capillary endothelial cells, whereas control cells didnot. Leung et al. (1989) Science, supra.

Although a vascular endothelial cell growth factor could be isolated andpurified from natural sources for subsequent therapeutic use, therelatively low concentrations of the protein in follicular cells and thehigh cost, both in terms of effort and expense, of recovering VEGFproved commercially unavailing. Accordingly, further efforts wereundertaken to clone and express VEGF via recombinant DNA techniques.(See, e.g., Ferrara, Laboratory Investigation 72:615-618 (1995), and thereferences cited therein).

VEGF is expressed in a variety of tissues as multiple homodimeric forms(121, 145, 165, 189, and 206 amino acids per monomer) resulting fromalternative RNA splicing. VEGF₁₂₁ is a soluble mitogen that does notbind heparin; the longer forms of VEGF bind heparin with progressivelyhigher affinity. The heparin-binding forms of VEGF can be cleaved in thecarboxy terminus by plasmin to release a diffusible form(s) of VEGF.Amino acid sequencing of the carboxy terminal peptide identified afterplasmin cleavage is Arg₁₁₀-Ala₁₁₁. Amino terminal “core” protein, VEGF(1-110) isolated as a homodimer, binds neutralizing monoclonalantibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1)and soluble forms of VEGF receptors with similar affinity compared tothe intact VEGF₁₆₅ homodimer.

Several molecules structurally related to VEGF have also beenidentified, including placenta growth factor (PIGF), VEGF-B, VEGF-C,VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra;Ogawa et al. J. Biological Chem. 273:31273-31281(1998); Meyer et al.EMBO J., 18:363-374(1999). A receptor tyrosine kinase, Flt-4 (VEGFR-3),has been identified as the receptor for VEGF-C and VEGF-D. Joukov et al.EMBO. J. 15:1751(1996); Lee et al. Proc. Natl. Acad. Sci. USA93:1988-1992(1996); Achen et al. (1998) Proc. Natl. Acad. Sci. USA95:548-553. VEGF-C has been shown to be involved in the regulation oflymphatic angiogenesis. Jeltsch et al. Science 276:1423-1425(1997).

Two VEGF receptors have been identified, Flt-1 (also called VEGFR-1) andKDR (also called VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; deVries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem.Biophys. Res. Commun. 187:1579-1586. Neuropilin-1 has been shown to be aselective VEGF receptor, able to bind the heparin-binding VEGF isoforms(Soker et al. (1998) Cell 92:735-45). Both Flt-I and KDR belong to thefamily of receptor tyrosine kinases (RTKs). The RTKs comprise a largefamily of transmembrane receptors with diverse biological activities. Atpresent, at least nineteen (19) distinct RTK subfamilies have beenidentified. The receptor tyrosine kinase (RTK) family includes receptorsthat are crucial for the growth and differentiation of a variety of celltypes (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57:433-478; Ullrichand Schlessinger (1990) Cell 61:243-254). The intrinsic function of RTKsis activated upon ligand binding, which results in phosphorylation ofthe receptor and multiple cellular substrates, and subsequently in avariety of cellular responses (Ullrich & Schlessinger (1990) Cell61:203-212). Thus, receptor tyrosine kinase mediated signal transductionis initiated by extracellular interaction with a specific growth factor(ligand), typically followed by receptor dimerization, stimulation ofthe intrinsic protein tyrosine kinase activity and receptortrans-phosphorylation. Binding sites are thereby created forintracellular signal transduction molecules and lead to the formation ofcomplexes with a spectrum of cytoplasmic signaling molecules thatfacilitate the appropriate cellular response. (e.g., cell division,differentiation, metabolic effects, changes in the extracellularmicroenvironment) see, Schlessinger and Ullrich (1992) Neuron 9:1-20.Structurally, both Flt-1 and KDR have seven immunoglobulin-like domainsin the extracellular domain, a single transmembrane region, and aconsensus tyrosine kinase sequence which is interrupted by akinase-insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.

(ii) Anti-VEGF Antibodies

Anti-VEGF antibodies that are useful in the methods of the inventioninclude any antibody, or antigen binding fragment thereof, that bindwith sufficient affinity and specificity to VEGF and can reduce orinhibit the biological activity of VEGF. An anti-VEGF antibody willusually not bind to other VEGF homologues such as VEGF-B or VEGF-C, norother growth factors such as PlGF, PDGF, or bFGF.

In certain embodiments of the invention, the anti-VEGF antibodiesinclude, but are not limited to, a monoclonal antibody that binds to thesame epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonalantibody generated according to Presta et al. (1997) Cancer Res.57:4593-4599. In one embodiment, the anti-VEGF antibody is “Bevacizumab(BV)”, also known as “rhuMAb VEGF” or “AVASTIN®”. It comprises mutatedhuman IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.

Bevacizumab and other humanized anti-VEGF antibodies are furtherdescribed in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005. Additionalantibodies include the G6 or B20 series antibodies (e.g., G6-31,B20-4.1), as described in PCT Publication No. WO2005/012359, PCTPublication No. WO2005/044853, and U.S. Patent Application 60/991,302,the content of these patent applications are expressly incorporatedherein by reference. For additional antibodies see U.S. Pat. Nos.7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332; WO 96/30046;WO94/10202; EP 0666868B1; U.S. Patent Application Publication Nos.2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and20050112126; and Popkov et al., Journal of Immunological Methods288:149-164 (2004). Other antibodies include those that bind to afunctional epitope on human VEGF comprising of residues F17, M18, D19,Y21, Y25, Q89, I91, K101, E103, and C104 or, alternatively, comprisingresidues F17, Y21, Q22, Y25, D63, 183 and Q89.

In one embodiment of the invention, the anti-VEGF antibody comprises aheavy chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 22) EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYFDVWGQGTLVT VSS.and a light chain variable region comprising the following amino acidsequence:

(SEQ ID NO: 23) DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYFTSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR.

In some embodiments the anti-VEGF antibody comprises a CDRH1 comprisingthe following amino acid sequence: GYTFTNYGMN (SEQ ID NO:24), a CDRH2comprising the following amino acid sequence: WINTYTGEPTYAADFKR (SEQ IDNO:25), a CDRH3 comprising the following amino acid sequence:YPHYYGSSHWYFDV (SEQ ID NO:26), a CDRL1 comprising the following aminoacid sequence: SASQDISNYLN (SEQ ID NO:27), a CDRL2 comprising thefollowing amino acid sequence: FTSSLHS (SEQ ID NO:28) and a CDRL3comprising the amino acid sequence: QQYSTVPWT (SEQ ID NO:29).

A “G6 series antibody” according to this invention, is an anti-VEGFantibody that is derived from a sequence of a G6 antibody or G6-derivedantibody according to any one of FIGS. 7, 24-26, and 34-35 of PCTPublication No. WO2005/012359, the entire disclosure of which isexpressly incorporated herein by reference. See also PCT Publication No.WO2005/044853, the entire disclosure of which is expressly incorporatedherein by reference. In one embodiment, the G6 series antibody binds toa functional epitope on human VEGF comprising residues F17, Y21, Q22,Y25, D63, I83 and Q89.

A “B20 series antibody” according to this invention is an anti-VEGFantibody that is derived from a sequence of the B20 antibody or aB20-derived antibody according to any one of FIGS. 27-29 of PCTPublication No. WO2005/012359, the entire disclosure of which isexpressly incorporated herein by reference. See also PCT Publication No.WO2005/044853, and U.S. Patent Application 60/991,302, the content ofthese patent applications are expressly incorporated herein byreference. In one embodiment, the B20 series antibody binds to afunctional epitope on human VEGF comprising residues F17, M18, D19, Y21,Y25, Q89, I91, K101, E103, and C104.

A “functional epitope” according to this invention refers to amino acidresidues of an antigen that contribute energetically to the binding ofan antibody. Mutation of any one of the energetically contributingresidues of the antigen (for example, mutation of wild-type VEGF byalanine or homolog mutation) will disrupt the binding of the antibodysuch that the relative affinity ratio (IC50mutant VEGF/IC50wild-typeVEGF) of the antibody will be greater than 5 (see Example 2 ofWO2005/012359). In one embodiment, the relative affinity ratio isdetermined by a solution binding phage displaying ELISA. Briefly,96-well Maxisorp immunoplates (NUNC) are coated overnight at 4° C. withan Fab form of the antibody to be tested at a concentration of 2 ug/mlin PBS, and blocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT) for 2 hat room temperature. Serial dilutions of phage displaying hVEGF alaninepoint mutants (residues 8-109 form) or wild type hVEGF (8-109) in PBTare first incubated on the Fab-coated plates for 15 min at roomtemperature, and the plates are washed with PBS, 0.05% Tween20 (PBST).The bound phage is detected with an anti-M13 monoclonal antibodyhorseradish peroxidase (Amersham Pharmacia) conjugate diluted 1:5000 inPBT, developed with 3,3′,5,5′-tetramethylbenzidine (TMB, Kirkegaard &Perry Labs, Gaithersburg, Md.) substrate for approximately 5 min,quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm.The ratio of IC50 values (IC50,ala/IC50,wt) represents the fold ofreduction in binding affinity (the relative binding affinity).

(iii) VEGF Receptor Molecules

The two best characterized VEGF receptors are VEGFR1 (also known asFlt-1) and VEGFR2 (also known as KDR and FLK-1 for the murine homolog).The specificity of each receptor for each VEGF family member varies butVEGF-A binds to both Flt-1 and KDR. The full length Flt-1 receptorincludes an extracellular domain that has seven Ig domains, atransmembrane domain, and an intracellular domain with tyrosine kinaseactivity. The extracellular domain is involved in the binding of VEGFand the intracellular domain is involved in signal transduction.

VEGF receptor molecules, or fragments thereof, that specifically bind toVEGF can be used in the methods of the invention to bind to andsequester the VEGF protein, thereby preventing it from signaling. Incertain embodiments, the VEGF receptor molecule, or VEGF bindingfragment thereof, is a soluble form, such as sFlt-1. A soluble form ofthe receptor exerts an inhibitory effect on the biological activity ofthe VEGF protein by binding to VEGF, thereby preventing it from bindingto its natural receptors present on the surface of target cells. Alsoincluded are VEGF receptor fusion proteins, examples of which aredescribed below.

A chimeric VEGF receptor protein is a receptor molecule having aminoacid sequences derived from at least two different proteins, at leastone of which is a VEGF receptor protein (e.g., the fit-1 or KDRreceptor), that is capable of binding to and inhibiting the biologicalactivity of VEGF. In certain embodiments, the chimeric VEGF receptorproteins of the invention consist of amino acid sequences derived fromonly two different VEGF receptor molecules; however, amino acidsequences comprising one, two, three, four, five, six, or all sevenIg-like domains from the extracellular ligand-binding region of thefit-1 and/or KDR receptor can be linked to amino acid sequences fromother unrelated proteins, for example, immunoglobulin sequences. Otheramino acid sequences to which Ig-like domains are combined will bereadily apparent to those of ordinary skill in the art. Examples ofchimeric VEGF receptor proteins include, e.g., soluble Flt-1/Fc, KDR/Fc,or FLt-1/KDR/Fc (also known as VEGF Trap). (See for example PCTApplication Publication No. WO97/44453)

A soluble VEGF receptor protein or chimeric VEGF receptor proteins ofthe invention includes VEGF receptor proteins which are not fixed to thesurface of cells via a transmembrane domain. As such, soluble forms ofthe VEGF receptor, including chimeric receptor proteins, while capableof binding to and inactivating VEGF, do not comprise a transmembranedomain and thus generally do not become associated with the cellmembrane of cells in which the molecule is expressed.

IV. Kits

In another aspect, provided is a kit comprising a PD-L1 axis bindingantagonist and/or a VEGF antagonist for treating or delaying progressionof a cancer in an individual or for enhancing immune function of anindividual having cancer. In some embodiments, the kit comprises a PD-1axis binding antagonist and a package insert comprising instructions forusing the PD-1 axis binding antagonist in combination with oxaliplatin,leucovorin, 5-FU with or without a VEGF antagonist to treat or delayprogression of cancer in an individual or to enhance immune function ofan individual having cancer. In some embodiments, the kit comprisesoxaliplatin, leucovorin, 5-FU with or without a VEGF antagonist and apackage insert comprising instructions for using the oxaliplatin,leucovorin, 5-FU with or without a VEGF antagonist in combination with aPD-1 axis binding antagonist to treat or delay progression of cancer inan individual or to enhance immune function of an individual havingcancer. In some embodiments, the kit comprises a PD-laxis bindingantagonist and oxaliplatin, leucovorin, 5-FU with or without a VEGFantagonist, and a package insert comprising instructions for using thePD-1 axis binding antagonist and the oxaliplatin, leucovorin, 5-FU withor without a VEGF antagonist to treat or delay progression of cancer inan individual or to enhance immune function of an individual havingcancer. Any of the PD-1 axis binding antagonists and/or VEGF antagonistsdescribed herein may be included in the kits.

EXAMPLES

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

Example 1: FOLFOX with or without Anti-VEGF Antibody Enhanced Anti-TumorActivity of Anti-PD-L1

To determine if FOLFOX (oxaliplatin, leucovorin and 5-fluorouracil) withor without anti-VEGF antibody enhanced the anti-tumor activity ofanti-PD-L1 mouse models of colorectal cancer were treated with thecombination treatments. Briefly, female C57BL/6 mice were inoculatedsubcutaneously in the unilateral thoracic region with 100,000 MC38murine colorectal cells in 100 microliters of HBSS:matrigel. When miceachieved a mean tumor volume of 220 mm³, they were randomly assigned toone of the treatment groups outlined below, at experimental day 0.Treatment was initiated on experimental day 1. Mice were weighed andtumors were measured 2-3 times per week for the duration of the study.

Experimental Groups:

-   -   1) Control (isotype control antibody (anti-gp120 antibody)), 10        mg/kg ip, 100 microliters, administered three times a week for        three weeks, n=10    -   2) anti-PD-L1 antibody, 10 mg/kg ip, 100 microliters,        administered three times a week for three weeks, n=10    -   3) FOLFOX (see below), administered once a week for two weeks,        n=10    -   4) FOLFOX (see below), administered once a week for two        weeks+anti-PD-L1 antibody, 10 mg/kg ip, 100 microliters,        administered three times a week for three weeks, n=10    -   5) FOLFOX (see below), administered once a week for two        weeks+anti-VEGF antibody, 5 mg/kg ip, 100 microliters,        administered two times a week for three weeks, n=10    -   6) FOLFOX (see below), administered once a week for two        weeks+anti-VEGF antibody, 5 mg/kg ip, 100 uL, administered two        times a week for three weeks+anti-PD-L1 antibody, 10 mg/kg ip,        100 microliters, administered three times a week for three        weeks, n=10        ip=intraperitoneally        sc=subcutaneously

For these studies, FOLFOX dosing was carried out as follows: onexperimental day 1 and experimental day 8, mice were administeredoxaliplatin, 5 mg/kg ip in 50 microliters of water immediately followedby leucovorin, 100 mg/kg ip in 250 microliters of water (administered attime=0 hour) and 5-FU, 25 mg/kg ip immediately followed by 5-FU, 25mg/kg sc (administered at time=2 hour). Anti-PD-L1 antibody andanti-gp120 antibody were dosed on experimental days 1, 3, 5, 8, 10, 12,15, 17, and 19 (administered at time=4 hours). Anti-VEGF antibody wasdosed on experimental day 1, 4, 8, 11, 15, 18 (administered at time=6hours).

Mice were monitored for tumor growth and body weight changes. Tumorvolumes were measured using UltraCal-IV calipers (Model 54-10-111; FredV. Fowler Company; Newton, Mass.). The following formula was used tocalculate tumor volume:Tumor Volume (mm³)=(Length×Width²)×0.5Length and width measurements were perpendicular to one another. Animalbody weights were measured using an Adventura Pro AV812 scale (OhausCorporation; Pine Brook, N.J.). Percent body weight change wascalculated using the following formula:Body weight change(%)=[(Weight_(Day new)−Weight_(Day))/Weight_(Day 0)]×100

Data were analyzed using R, version 2.9.2 (R Development Core Team 2008;R Foundation for Statistical Computing; Vienna, Austria), and the mixedmodels were fit within R using the nlme package, version 3.1-96(Pinheiro et al. 2009). Plotting was performed in Prism, version 5.0bfor Mac (GraphPad Software, Inc.; La Jolla, Calif.).

A mixed modeling approach was used to analyze the repeated measurementof tumor volumes from the same animals over time (Pinheiro and Bates2000). This approach addresses both repeated measurements and modestdropouts before study end for reasons classifiable statistically asmissing at random (MAR). The fixed effect changes in log₂(volume) bytime and dose are modeled as the sum of the main effects and interactionof a natural cubic regression spline basis in time with anauto-determined natural spline basis in dose. Intercepts and growthrates (slopes) were assumed to vary randomly by animal. Tumor growthinhibition as a percentage of the control-treated group (% TGI) wascalculated as the percentage of the area under the fitted curve (AUC)for the respective treatment group per day in relation to the controlwhile the control treated mice were still on study, using the followingformula:% TGI=100×(1−AUC_(dose)/AUC_(vehicles))

For these studies, Complete Response (CR) was defined as an individualanimal whose tumor volume fell below the Limit of Detection (LOD), atany time during the study. Partial Response (PR) was defined as anindividual animal whose tumor volume decreased by 50% of its initialtumor volume at any time during the study. Overall Response Rate (ORR)was defined as the sum of the complete and partial responses. Time ToProgression 5× (TTP5×) was defined as the time in days for a group'sfitted tumor volume (based upon the mixed modeling analysis describedabove) to exceed 5 times the starting volume, rounded to the nearesthalf day and reported as the TTP5× for that group. Linear mixed-effectsanalysis was also employed to analyze the repeated measurement of bodyweight changes from the same animals over time.

Blockade of the PD-1 axis using anti-PD-L1 antibody was effective as asingle agent therapy at preventing tumor growth. Combination treatmentof anti-PD-L1 antibodies with oxaliplatin, leucovorin and 5-FU (FOLFOX)significantly inhibited tumor growth indicating that this chemotherapycombination enhanced the anti-tumor activity of anti-PD-L1 antibodies(FIG. 1). Addition of anti-VEGF to this combination treatment furtherenhanced this anti-tumor activity and as well as the durability of theanti-tumor response even after the cessation of treatment (FIG. 4).

Example 2: A Phase 1b Study of MPDL3280A with Bevacizumab with orwithout Modified FOLFOX-6

The primary aim of the study is to assess the safety, pharmacology andpreliminary efficacy of MPDL3280A administered with bevacizumab (Arm A)and with bevacizumab plus FOLFOX (specifically, modified FOLFOX-6, ormFOLFOX-6; Arm B) in patients with solid tumors including metastaticcolorectal cancer (mCRC). Arm A will evaluate MPDL3280A at 10 mg/kg (ora selected dose level not to exceed the single-agent MTD or MAD) withbevacizumab (15 mg/kg) on an every-3-week (q3w) schedule for up to oneyear. Patients who have not received oxaliplatin for metastatic diseasewill be enrolled in Arm B to receive MPDL3280A with bevacizumab andFOLFOX on an every-2-week (q2w) schedule. mFOLFOX-6 regimen consist ofthe following: oxaliplatin (85 mg/m²) administered intravenously (IV)concurrently with leucovorin (400 mg/m²) administered IV over about 120minutes followed by 5-FU (400 mg/m2) administered as an IV bolus,followed by 2400 mg/m² administered by continuous IV infusion over about46 hours. Oxaliplatin will be administered for up to eight cycles.Treatment may be continued for up to one year.

What is claimed is:
 1. A method for treating or delaying progression ofcolorectal cancer in an individual comprising administering to theindividual an effective amount of an anti-PD-L1 antibody, oxaliplatin,leucovorin and 5-FU, wherein the method further comprises administeringbevacizumab, wherein the anti-PD-L1 antibody comprises a heavy chainvariable region and a light chain variable region, wherein: (a) theheavy chain variable region comprises an HVR-H1, HVR-H2 and HVR-H3, andwherein: (i) the HVR-H1 comprises the amino acid sequence of SEQ ID NO:15; (ii) the HVR-H2 comprises the amino acid sequence of SEQ ID NO: 16;(iii) the HVR-H3 comprises the amino acid sequence of SEQ ID NO: 3; and(b) the light chain variable region comprises an HVR-L1, HVR-L2 andHVR-L3, and wherein: (iv) the HVR-L1 comprises the amino acid sequenceof SEQ ID NO: 17; (v) the HVR-L2 comprises the amino acid sequence ofSEQ ID NO: 18; and (vi) the HVR-L3 comprises the amino acid sequence ofSEQ ID NO:
 19. 2. The method of claim 1, wherein the anti-PD-L1 antibodyis a monoclonal antibody.
 3. The method of claim 1, wherein theanti-PD-L1 antibody is an antibody fragment selected from the groupconsisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂ fragments.
 4. Themethod of claim 1, wherein the anti-PD-L1 antibody is a humanizedantibody.
 5. The method of claim 1, wherein the anti-PD-L1 antibodycomprises a heavy chain variable region and a light chain variableregion, wherein: (a) the heavy chain variable region amino acid sequencehas at least 90% sequence identity to the heavy chain variable regionamino acid sequence of SEQ ID NO:20, and (b) the light chain variableregion amino acid sequence has at least 90% sequence identity to thelight chain variable region amino acid sequence of SEQ ID NO:21.
 6. Themethod of claim 5, wherein: (a) the heavy chain variable region aminoacid sequence has at least 95% sequence identity to the heavy chainvariable region amino acid sequence of SEQ ID NO:20, and (b) the lightchain variable region amino acid sequence has at least 95% sequenceidentity to the light chain variable region amino acid sequence of SEQID NO:21.
 7. The method of claim 6, wherein: (a) the heavy chainvariable region amino acid sequence has at least 99% sequence identityto the heavy chain variable region amino acid sequence of SEQ ID NO:20,and (b) the light chain variable region amino acid sequence has at least99% sequence identity to the light chain variable region amino acidsequence of SEQ ID NO:21.
 8. The method of claim 7, wherein (a) theheavy chain variable region amino acid sequence has at least 99%sequence identity to the heavy chain variable region amino acid sequenceof SEQ ID NO:20, and (b) the light chain variable region comprises theamino acid sequence of SEQ ID NO:21.
 9. The method of claim 8, whereinthe anti-PD-L1 antibody further comprises a human IgG1 constant region.10. The method of claim 9, wherein the anti-PD-L1 antibody comprises aneffector-less Fc mutation, wherein the effector-less Fc mutation isN297A.